LOCUS       sequence-165864-        6314 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of recombinant human mature (delta19) ETFalpha wt without
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6314)
  AUTHORS   Malecki J, Ho AY, Moen A, Dahl HA, Falnes PO
  TITLE     Human METTL20 is a mitochondrial lysine methyltransferase that 
            targets the beta subunit of electron transfer flavoprotein (ETFbeta)
            and modulates its activity.
  JOURNAL   J Biol Chem. 2015 Jan 2;290(1):423-34. doi: 10.1074/jbc.M114.614115.
            Epub 2014 Nov 21.
  PUBMED    25416781
REFERENCE   2  (bases 1 to 6314)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.M114.614115"; journalName: "J Biol Chem"; date: 
            "2015-01-2- 2"; volume: "290"; issue: "1"; pages: "423-34"
COMMENT     Sequence Label: pF710-pETDuet-1-Hs-delta19-ETFalpha-noHis wt
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             1260..1304
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1342..1360)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1356..1403
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1440..1895
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1527..1546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1737..1758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2010..2801,2802..2870))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2633..2652
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2871..2963)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3044..3632
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3533..3552
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3786..3803
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3818..3960
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3886..3905)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4046..4068
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4062..4253)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4825..5907)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5869..5888
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5908..5985)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6191..6210
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6298..6314,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgcagagtac cctggtaata gctgagcatg caaatgattc cctagcaccc attactttaa
      361 ataccattac tgcagccaca cgccttggag gtgaagtgtc ctgcttagta gctggaacca
      421 aatgtgacaa ggtggcacaa gatctctgta aagtagcagg catagcaaaa gttctggtgg
      481 ctcagcatga tgtgtacaaa ggcctacttc cagaggaact gacaccattg attttggcaa
      541 ctcagaagca gttcaattac acacacatct gtgctggagc atctgccttc ggaaagaacc
      601 ttttgcccag agtagcagcc aaacttgagg ttgccccgat ttctgacatc attgcaatca
      661 agtcacctga cacatttgtg agaactattt atgcaggaaa tgctctatgt acagtgaagt
      721 gtgatgagaa agtgaaagtg ttttctgtcc gtggaacatc ctttgatgct gcagcaacaa
      781 gtggcggtag tgccagttca gaaaaggcat caagtacttc accagtggaa atatcagagt
      841 ggcttgacca gaaattaaca aaaagtgatc gaccagagct aacaggtgcc aaagtggtgg
      901 tatctggtgg tcgaggcttg aagagtggag agaactttaa gttgttatat gacttggcag
      961 atcaactaca tgctgcagtt ggtgcttccc gtgctgctgt tgatgctggc tttgttccca
     1021 atgacatgca agttggacag acgggaaaaa tagtagcacc agaactttat attgctgttg
     1081 gaatatctgg agccatccaa catttagctg ggatgaaaga cagcaagaca attgtggcaa
     1141 ttaataaaga cccagaagct ccaattttcc aagtggcaga ttatggaata gttgcagatt
     1201 tatttaaggt agttcctgaa atgactgaga tattgaagaa aaaatgactc gagtctggta
     1261 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     1321 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1381 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1441 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1501 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1561 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1621 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1681 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1741 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1801 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1861 cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta
     1921 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1981 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2041 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2101 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2161 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2221 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2281 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2341 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2401 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2461 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2521 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2581 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2641 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2701 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2761 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2821 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     2881 tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2941 atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt
     3001 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3061 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3121 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3181 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3241 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3301 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3361 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3421 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3481 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3541 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3601 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3661 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3721 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3781 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3841 cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga
     3901 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3961 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4021 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4081 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     4141 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4201 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4261 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4321 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4381 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4441 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4501 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4561 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4621 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4681 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     4741 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4801 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4861 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4921 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4981 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5041 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5101 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5161 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5221 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5281 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5341 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5401 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5461 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5521 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5581 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5641 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5701 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5761 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5821 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5881 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5941 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     6001 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6061 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6121 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6181 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6241 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaattaa
     6301 tacgactcac tata
//
